Human keratinocyte (HK) motility plays an important role in the re-epithelialization of human skin wounds. Extracellular matrices (ECMs) and serum growth factors (GFs) are the two main stimuli that control HK migration. ECMs and GFs bind to their cognate receptors and activate distinct, yet overlapping, signaling networks. The SPECIFIC function of ECMs versus GFs in the control of HK migration remains unclear, Our preliminary studies indicate that ECMs, but not GFs, initiate migration. The ECM-induced migration, however, is partial and non-directional, while GFs' role appears to direct and optimize migration. Our hypothesis is that HK migration on connective tissue is initiated by ECMs, which work in concert with GFs to optimize the molility and provide directionality. Here, we propose to study the signaling mechanisms of ECM-initiated random motility and growth factor-optimized directional motility. The focus of these studies will be on the signal transduction by a collagen matrix without GFs (random migration) and by a collagen matrix plus GFs (directional and optimal migration). Specifically, we will: 1) study the role of the Rho family GTPases in HK migration by a collagen matrix in the presence or absence of GFs. Different Rho family GTPase members have distinct functions in the regulation of the actin cytoskeleton and cell migration. Their functions in HK motility are not clear. Both pharmacological and genetic approaches will be used to study these GTPases in HKs; 2) study how p38-MAPK and PKC-delta regulate random versus directional migration. Our recent findings show that p38-MAPK and PKC-delta are independently required for HK motility on a collagen matrix (in press). Here, we will further investigate the specific function of these two pathways in the integrin and the growth factor receptor signaling in HKs; 3) identify and characterize HK migration-linked genes by a novel "TGF-beta block" approach. We will take advantage of the fact that TGF-beta blocks proliferation but not migration in HKs. cRNAs from TGF-beta- treated 1) non-migrating HKs, 2) randomly migrating HKs, and 3) directionally migrating HKs will be subjected to DNA microarray analysis. The microarray-identified genes will be further subjected to a "pathway-screening" approach to narrow down the Rac1-p38-MAPK pathway- and the PKC-delta pathway-induced genes. These studies collectively will shed new light on the molecular mechanisms of wound re-epithelialization.